title: Development of a high-throughput human cytomegalovirus quantitative PCR cell-based assay.
abstract: This report describes the development and optimization of a quantitative real-time PCR assay for evaluating human cytomegalovirus (CMV) replication in vitro and susceptibility to antiviral drugs. This assay measures the level of intracellular CMV DNA in both 96- and 384-well microplate formats. Normalization of CMV levels using mitochondrial DNA enhanced the robustness of the assay and minimized variability. The assay throughput was further enhanced by eliminating several wash steps and by lysing the cells directly in the presence of cell culture media, both of which had no impact on the assay metrics. The assay was validated using several known CMV antiviral compounds. The CMV quantitative PCR (qPCR) assay represents a rapid, reliable and reproducible method that can be used with both CMV laboratory strains and clinical isolates.
authors.affiliation: Boehringer Ingelheim (Canada) Ltd., Research & Development, Department of Biological Sciences, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada.
authors.forename: Sonia
authors.initials: S
authors.lastname: Tremblay
authors.forename: Nathalie
authors.initials: N
authors.lastname: Dansereau
authors.forename: Scott
authors.initials: S
authors.lastname: Balsitis
authors.forename: Michael
authors.initials: M
authors.lastname: Franti
authors.forename: Louie
authors.initials: L
authors.lastname: Lamorte
mesh_terms: D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D003600:Cytosol / Q000821:virology; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D013997:Time Factors; D014773:Virology / Q000379:methods* / Q000592:standards; D014779:Virus Replication
chemical_list: D004272:DNA, Mitochondrial; D004279:DNA, Viral
keywords: Antiviral drug; Clinical isolates; Cytomegalovirus; Quantitative PCR; Real-time PCR; Replication